HIV infection of primary human cells typically requires interaction with both the primary receptor CD4 and a chemokine coreceptor, either CCR5 or CXCR4. There may be additional cellular factors involved in determining the efficiency of HIV entry, however, which may explain (in part) the marked differences in susceptibility to HIV infection among primary cells from different donors. During studies to identify H1V cofactors in macrophages (Mphi), we found that four independently generated monoclonal antibodies (mAb) to a tetraspan membrane glycoprotein, CD63, could block HIV entry into Mphi, but not T-cells. The function of CD63 is not well understood, but another tetraspan protein, CD81, has been proposed as a receptor for Hepatitis C virus, and CD9 has been implicated in infection by feline immunodeficiency virus (FIV). CD63 has previously been recognized as a platelet activation marker, as well as a marker for malignant melanoma, both seemingly unrelated to retroviral infection. Our experiments will investigate the role of CD63 in HIV entry into MO). These studies may lead to development of novel antiretroviral therapies targeting HIV entry. We will pursue these Aims over four years: Specific Aim 1. To test the hypothesis that CD63 has a specific role in facilitating HIV entry into Mphi, we will identify potential mechanisms of CD63-mediated HIV entry. Experiments will be prioritized based on initial studies that more precisely identify the step of HIV entry involved in anti-CD63 inhibition, directing initial studies toward late entry events, such as the role of CD63 in CD4 turnover or virus/receptor endocytosis and signal transduction, or early events, such as anti-CD63 interactions at the cell surface including binding and CCR5-dependent entry pathways. Specific Aim 2. To test the hypothesis that cell-specific differences between HIV target cells affect the role of CD63 in facilitating HIV entry. We will control for and test effects of various levels of receptor/coreceptor/CD63 expression and activation markers on susceptibility to anti-CD63 inhibition in primary CD4+ cells. We will also test cell line models of anti-CD63 inhibition, which would facilitate cell manipulation and the consistency of inhibitory effects in these studies. Specific Aim 3. To test the hypothesis that infection of Mphi by diverse primary HIV-1 strains can be inhibited by anti- CD63 mAb. This will include both subtype B and non-subtype B strains, including some that exclusively use CCR5 as a coreceptor for efficient infection (R5), as well as dual-tropic strains that use either CXCR4 or CCR5 (R5X4), and X4 strains able to replicate in Me. Achieving these Aims will provide important new insights into the mechanism of HIV-1 entry, and may well provide important new targets for antiretroviraI therapy.